PURPOSE: To investigate the behavior of perflubutane microbubbles outside and inside Kupffer cells during diagnostic ultrasound (US) examination, and to determine the thresholds of the acoustic pressure of different kinds of behavior. METHODS: Acoustic behavior of perflubutane microbubbles inside and outside Kupffer cells in an acoustic field induced by a clinical US transducer and equipment was optically observed in vitro. The acoustic pressure was measured simultaneously by a calibrated hydrophone and an oscilloscope. RESULTS: The acoustic behavior of microbubbles was optically categorized as stabilization, oscillation, transposition, shrinkage, and destruction. The mechanical index (MI) displayed on the US equipment correlated well with the acoustic pressure at the level of microbubbles measured hydrophonically. At a frame rate of 15 Hz with a frequency of 3.5 MHz and pulse repetition frequency of 3 KHz, the thresholds in term of MI for free microbubbles to begin oscillation, reach best oscillation, transposition, shrinkage, and destruction were 0.21, 0.44, 0.53, 0.75, and 1.03, respectively. Although adherent and phagocytosed microbubbles showed more stability enduring insonation compared with free microbubbles, the thresholds of shrinkage and destruction were MI 1.03 and 1.18 for adherent microbubbles, and 1.18 and 1.37 for phagocytosed microbubbles, respectively. Neither oscillation nor transposition of microbubbles inside Kupffer cells was observed microscopically. No cell damage because of microbubbles destruction was found in the present study. CONCLUSION: Perflubutane microbubbles outside and inside Kupffer cells respond to external US insonation with same parameters of a clinical contrast-enhanced US study according to the acoustic pressure. Free microbubbles behave as stabilization, oscillation, transposition, shrinkage, and destruction under insonation. The adherent and phagocytosed microbubbles are more stable under insonation than free microbubbles, but still respond showing shrinkage and destruction when MI is over 1.03.